Immuno-based botulinum toxin serotype A activity assays
The present specification discloses SNAP-25 compositions, methods of making α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P 1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, α-SNAP-25 antibodies that bind an epitope comprising a carboxyl-terminus at the P 1 residue from the BoNT/A cleavage site scissile bond from a SNAP-25 cleavage product, methods of detecting BoNT/A activity, and methods of detecting neutralizing α-BoNT/A antibodies.
1. A differentiated mammalian neuroblastoma cell susceptible to intoxication by about 10 pM or less of a botulinum neurotoxin serotype A (BoNT/A), wherein the susceptibility to intoxication is the lower limit of quantitation of cleaved BoNT/A substrate as determined by Western blot.
2. The differentiated mammalian neuroblastoma cell of claim 1 , wherein the cell is a differentiated SiMa cell or a differentiated Neuro-2a cell.
3. The differentiated mammalian neuroblastoma cell of claim 1 , wherein the cell is susceptible to intoxication by about 1 pM or less of BoNT/A.
4. The differentiated mammalian neuroblastoma cell of claim 1 , wherein the cell comprises a non-naturally occurring Synaptosomal-Associated Protein of 25 kDa (SNAP-25).
5. The differentiated mammalian neuroblastoma cell of claim 4 , wherein the non-naturally occurring SNAP-25 comprises a fluorophore.
6. The differentiated mammalian neuroblastoma cell of claim 1 , wherein the susceptibility to intoxication is measured by disappearance of an intact BoNT/A substrate.
7. The differentiated mammalian neuroblastoma cell of claim 6 , wherein the intact BoNT/A substrate is a non-naturally occurring SNAP-25.
8. The differentiated mammalian neuroblastoma cell of claim 6 , wherein the susceptibility to intoxication is measured following incubation of the cell in serum-free media for between about 24 and about 72 hours.
9. The differentiated mammalian neuroblastoma cell of claim 8 , wherein BoNT/A is present in the serum-free media for between about 24 to about 72 hours.
10. The differentiated mammalian neuroblastoma cell of claim 1 , wherein the Western blot is performed using S9684 α-SNAP-25 rabbit polyclonal antiserum as primary detection antibody.
11. A Neuro-2a cell susceptible to intoxication by about 10 pM or less of a BoNT/A, wherein the susceptibility to intoxication is the lower limit of quantitation of cleaved BoNT/A substrate as determined by Western blot following incubation of the cell in serum-free media for between about 24 hours and about 72 hours.
12. The Neuro-2a cell of claim 11 , wherein BoNT/A is present in the serum-free media for between about 24 hours and about 72 hours.
13. The Neuro-2a cell of claim 11 , wherein the cell comprises a non-naturally occurring SNAP-25.
14. The Neuro-2a cell of claim 13 , wherein the non-naturally occurring SNAP-25 comprises a fluorophore.
15. The Neuro-2a cell of claim 11 , wherein the Western blot is performed using S9684 α-SNAP-25 rabbit polyclonal antiserum as primary detection antibody.
16. The Neuro-2a cell of claim 11 , wherein the cell is susceptible to intoxication by about 1 pM or less of BoNT/A.